Synthesis of novel methyl jasmonate derivatives and evaluation of their biological activity in various cancer cell lines

Article abstract of DOI:10.1016/j.bioorg.2019.103146

Warburg hypothesized that the energy consumption of cancer cells is different than the normal cells. When compared to normal conditions, cancer cells do not undergo tricarboxylic acid (TCA) cycle therefore resulting in more lactate in the cells. Glycolysis pathway is a way of cancer cells to provide energy. The first step in glycolysis is the phosphorylation of glucose to glucose-6-phosphate. This reaction is catalyzed by the hexokinase-II enzyme (HK-II) which is known to be overexpressed in tumor cells. The feeding of cancer cells can be prevented by inhibiting the hexokinase-II enzyme in the first step of aerobic glycolysis. In literature, Methyl Jasmonate (MJ) is known as a Hexokinase-II inhibitor since it disposes VDAC and HK-II interaction on mitochondrial membrane. In our study, we aimed to increase the activity by synthesizing the novel MJ analogues with appropriate modifications. Here we report Hexokinase-2 enzyme and cell viability study results in different cancer cells. Based on the three different cancer cell lines we investigated, our novel MJ analogues proved to be more potent than the original molecule. Thus this research may provide more efficacious/novel HK-II inhibitors and may shed light to develop new anti-cancer agents.

Full text of DOI:10.1016/j.bioorg.2019.103146

Accepted Manuscript
Synthesis of Novel Methyl Jasmonate Derivatives and Evaluation of Their Bi-
ological Activity in Various Cancer Cell Lines
Bilgesu Onur Sucu, Ozgecan Savlug Ipek, Sukran Ozdatli Kurtulus, Busra
Emine Yazici, Nihal Karakas, Mustafa Guzel
PII:
S0045-2068(19)30630-3
https://doi.org/10.1016/j.bioorg.2019.103146
103146
DOI:
Article Number:
Reference:
YBIOO 103146
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
24 April 2019
19 July 2019
22 July 2019
Please cite this article as: B. Onur Sucu, O. Savlug Ipek, S. Ozdatli Kurtulus, B. Emine Yazici, N. Karakas, M.
Guzel, Synthesis of Novel Methyl Jasmonate Derivatives and Evaluation of Their Biological Activity in Various
Cancer Cell Lines, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103146
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Synthesis of Novel Methyl Jasmonate Derivatives and Evaluation of Their
Biological Activity in Various Cancer Cell Lines
a,b
b,c
b
d
b,d
Bilgesu Onur Sucu , Ozgecan Savlug Ipek , Sukran Ozdatli Kurtulus , Busra Emine Yazici , Nihal Karakas and
Mustafa Guzel
*b,e
a
Istanbul Medipol University, Vocational School of Health Services, Pharmacy Services, Kavacik Campus, Kavacik-
Beykoz/ISTANBUL 34810, Turkey.
Istanbul Medipol University, Regenerative and Restorative Medicine Research Center (REMER), Kavacik Campus, Kavacik-
b
Beykoz/ISTANBUL 34810, Turkey.
c
Yildiz Technical University, Graduate School Of Natural And Applied Sciences, Department of Chemistry, Besiktas/ISTANBUL,
3
4349, Turkey.
d
Istanbul Medipol University, School of Medicine, Department of Medical Biology, Kavacik Campus, Kavacik-Beykoz/ISTANBUL
4810, Turkey.
Istanbul Medipol University, International School of Medicine, Department of Medical Pharmacology, Kavacik Campus,
3
e
Kavacik-Beykoz/ISTANBUL 34810, Turkey.
Keywords: Cancer Therapy, Aerobic Glycolysis, Warburg Effect, Hexokinase-II Inhibition, Methyl Jasmonate, Novel Drug
Discovery and Development.
ABSTRACT
Warburg hypothesized that the energy consumption of cancer cells is different than the normal cells. When compared to normal
conditions, cancer cells do not undergo tricarboxylic acid (TCA) cycle therefore resulting in more lactate in the cells. Glycolysis
pathway is a way of cancer cells to provide energy. The first step in glycolysis is the phosphorylation of glucose to glucose-6-
phosphate. This reaction is catalyzed by the hexokinase-II enzyme (HK-II) which is known to be overexpressed in tumor cells.
The feeding of cancer cells can be prevented by inhibiting the hexokinase-II enzyme in the first step of aerobic glycolysis. In
literature, Methyl Jasmonate (MJ) is known as a Hexokinase-II inhibitor since it disposes VDAC and HK-II interaction on
mitochondrial membrane. In our study, we aimed to increase the activity by synthesizing the novel MJ analogues with
appropriate modifications. Here we report Hexokinase-2 enzyme and cell viability study results in different cancer cells. Based
on the three different cancer cell lines we investigated, our novel MJ analogues proved to be more potent than the original
molecule. Thus this research may provide more efficacious/novel HK-II inhibitors and may shed light to develop new anti-cancer
agents.
1. Introduction
Glycolysis, which is utilized by organisms that perform cellular respiration, is the first step in the breakdown of glucose to
obtain energy (ATP) for the cellular metabolism. In addition to this, glycolysis does not occur only in the presence of oxygen, a
lot of anaerobic organisms also have glycolytic metabolism of glucose. This pathway has almost 10 steps that give adenosine
triphosphate (ATP), NADH, pyruvate molecules and etc. These intermediates are necessary for synthesis of other cellular
constituents [1]. Cancer and normal cells depart from each other with regard to morphology and metabolic conduct. Normal
cells mainly obtain their source of energy by oxidative phosphorylation in mitochondria. Otto Warburg proposed that cancer cells
prefer a high rate of glycolytic pathway even in the presence of abundant oxygen for their energy production rather than the
tricarboxylic acid (TCA) cycle. This metabolic state involving increased glucose uptake leads to local acidification through
enhanced lactate production.This hypothesis is verified with many studies in literature [2]. The first step of glycolytic pathway is
phosphorylation of glucose to glucose-6-phosphate (G-6-P) via phosphate transfer from ATP. This reaction is catalyzed with
Hexokinase (HK) enzyme [3]. There are four HK isozymes categorized as HK-I, II, III and IV. These isozymes found in different
regions in the mammalian tissues. HK-I generally occupied in most tissues; on a vast scale in brain, kidney, and red blood cells
(RBCs). Hexokinase II is high abundance in the cardiac and skeletal muscle in a healthy tissue. In all tissues HK-III isozyme is
evenly distrubitued, despite of less existing percentage amount when compared to other isozymes . Hexokinase-IV, which is
also known as glucokinase (GK), is predominantly located in liver, pancreas and intestine. All of these HK isozymes are
important in cellular metabolism and play crucial role in major cellular events [4]. According to literature, the expression level of
HK-II in cancer cells is much higher than in normal cells [3]. It has been postulated that Mitochondrial Outer Membrane (MOM)
via interaction with the voltage-dependent anion channel (VDAC) is balanced by HK-II to prevent apoptosis. In tumors the HK-
II gene is elevated and HK-II RNA level is overexpressed [5]. This isozyme is a dominant in insulin-responsive tissues such as
heart, skeletal muscle, adipose tissue and various types of cancers, such as ovarian, gastric, breast, cervical carcinoma.
Moreover, HK-II is upregulated in many types of tumors by enhanced aerobic glycolysis in tumor cells [6]. Notably, these
important affects of HK-II show that inhibition of this enyzme can be an efffective and attractive target for anticancer drug
development.
Based on this information we thought that potent and efficacious new molecules that target the selective inhibition of this
isozyme can provide good potential candidates as anti-cancer agents. In literature, currently four clinical and pre-clinical
candidates have been reported as small molecule HK-II inhibitors which are Methyl Jasmonate (MJ), 3-Bromopyruvate (3-BP),
2-deoxyglucose (2-DG), and Lonidamine (Figure 1).

O
Figure 1. Small molecule Hexokinase-II inhibitors known in
literature.
OH
O
O
O
OH
HO
HO
N
Br
O
N
Recently, there has been many reports that MJ exhibits
reasonably good anti-cancer activity in vitro and in vivo
experiments. The previous studies demonstrated that MJ induces
suppression of cellular proliferation and death in diverse human
and mouse cancer cell lines, involving breast, prostate,
O
OH
O
Cl
O
Cl
MJ
3-PA
2-DG
Lonidamine
melanoma, lymphoblastic leukemia, and lymphoma cells [7]. Moreover important anti-inflammatory activity was also reported
for MJ analogues that showed increased activity than natural anti-inflammatory prostaglandins. These results have triggered
much research interest in jasmonates as class of versatile bioactive molecules [8]. In the light of these findings and based on
literature search we have designed handful of novel MJ analogues that has never been synthesized before and investigated
their anti-cancer activity in various cancer cell lines. Thus, in the present study we report that the synthesis, enzyme activities
and cell viabilities of novel methyl jasmonate analogues.
2. Results and discussion
2.1. Synthetic chemistry
Initially we started modifiying the ester portion of MJ by installing druggable handles such as oxodiazole moieties. Therefore,
we started from jasmonic acid (JA) from which was obtained initial building block pseudo ester with DCC coupling strategy
followed by basic condensation provided novel 1,2,4- oxadiazoles 1 and 2. In order to obtain 1,3,4-oxadiazoles we started with
3
hydrazide formation from JA, followed by Schotten-Baumann reaction with 4-chlorobenzoyl chloride and POCl condensation
yielded us desired molecule 5 (Scheme 1).
Scheme 1. Synthesis of Methyl Jasmonate Analogues 1-5a
O
O
O
OH
a
b
c
O
O
N
N
OH
O
R1
O
O
N
N
N
R1
R1
H2N
1
2
CH3
3
JA
d
O
O
O
e
f
O
N
N
O
NH
O
O
NH
HN
H2N
Cl
Cl
4
5
aReagents and conditions: (a) For 1: DCC, acetamide oxime, DMF, rt. For 2: DCC, benzamide oxime, acetone, rt. (b) For 1:
DCC, DMF, 115 ° C. For 2: KOH, DMSO, rt. (c) NaBH , MeOH (d) hydrazine monohydrate, 80 ° C, 3h. (e) 4-chlorobenzoyl
chloride, ACN/H O, 0 ° C. (f) POCl , toluene, 110 ° C, 16h.
4
2
3
Based on the initial biological findings and cell based assay results (Table 1), among one of the synthesized more active
oxadiazole (1) (IC50=0.40 M) molecule was subjected to reduction procedure to obtain compound 3 (IC50=0.27 M) which
demonstrated much more activity.
After obtaining positive results we then decided to synthesize various amide analogues (6 and 7) as well as modifying it to
methyl carbamate (9). Amides were routinely obtained via HBTU coupling procedure as shown Scheme 2. Compound 9 was
synthesized from an isocyanate intermediate (8) using Curtius rearrangement procedure. Overall these amides showed
reasonable enzyme inhibition activity, however cell based results did not translate the same activity.
Scheme 2. Synthesis of Methyl Jasmonate Analogues 6-9a

O
O
a
R2
6
7
O
O
O
N
OH
NH
HO
R2
O
JA
OH
HO OH
b
O
O
c
N
C
HN
O
O
O
8
9
aReagents and conditions: (a) HBTU, TEA, (2-aminoethyl)morpholine (for 6), D-(+)-Glucosamine hydrochloride (for 7), (b)
DMAP, DPPA, TEA, toluene, 110 ° C. (c) NaOMe, MeOH, rt.
We then turned our attention to reversed esters and fused molecules. Therefore, after reduction of the ester to its
corresponding primary alcohol (10), two heteroatom containing aromotic esters (13 and 14) were obtained via CDI coupling
procedure. In order to modify the ketone portion of MJ, we formylated the --carbon of the ketone followed by phenyl hydrazine
addition afforded the fused phenyl-pyrazole 12 (Scheme 3). The preliminary enzyme assay results of these novel analogues
when compared to initial series (compounds 1-3) surprisingly did not provide better activity, however there may need to be more
reversed ester analogues synthesized in order to see the clear evidence in inferiority and observe solid structural activity
relationship (SAR).
Scheme 3. Synthesis of Methyl Jasmonate Analogues 10-14a
OH
O
O
O
a
b
O
O
OH
OH
10
11
d
c
O
N
N
R3
O
1
3
4
O
N
O
R3
OH
N
1
S
12
aReagents and conditions: (a) TMS, NaI, TEA, ACN 40 °C, 1.5 h. (b) NaOMe, ethyl formate, toluene, rt. (c),phenyl hydrazine,
MeOH. (d) : NaH, DMF, CDI-activated carboxylic acid 0 °C→rt.
Our strategy to modify the ketone portion continued with reductive amination followed by synthesizing novel urea analogues
3
17 and 18. We first converted MJ to its corresponding amines via reductive amination procedure using Na(OAc) BH to obtain
the desired secondary amines which were transformed to urea analogues with isocyanates (17 and 18). Our observation for the
urea analogues is that the elongation of the MJ on ketone side did not seem to increase activity (IC50=38.14 and 14.0 µM
respectively) since the molecular weight increased dramatically. This can be hypothesized that increasing the bulk in ketone
portion of MJ somehow changes the binding mode of these molecules with HK-II enyzme. We are still investigating the binding
mode of these novel analogues and will be reported in due course after verification.

Scheme 4. Synthesis of Methyl Jasmonate Analogues 15-18a
R4
17
O
R4
HN
O
N
O
N
18
c
R4
O
NH
O
a,b
O
17
O
O
O
O
d
R4
N
O
H
N
R4
N
N
H
O
O
O
O
O
18
aReagents and conditions: (a) Amine, ACN, 2h. (b) Na(OAc)
diisocyanate, DCM, rt
BH, overnight. (c) 2-metoxyphenyl isocyanate, DCM, rt. (d) 1,4-
3
We then pursued much challenging modifications on MJ which is shown on Scheme 5. Our aim was to displace the alkyl side
chain of MJ with an appropriate substituents or aromotic structures. Starting with 2-cyclopenten-1-one, -bromination, Suzuki
coupling and Michael addion reactions provided compound 21 with overall good yield. Biological activity of this molecule did not
make much difference when compared to other modifications, which may suggest that the side chain modifications do not
improve the potency. These findings led us investigate more options on ester and ketone part of MJ.
Scheme 5. Synthesis of Methyl Jasmonate Analogues 19-21a
O
O
O
O
a
Br
b
c
O
O
19
20
O
O
21
a
®
2 3 3 4 2
Reagents and conditions: (a) OXONE , DCM, HBr, TEA, rt, 4h. (b) Phenyl boronic acid, Na CO , Pd(PPh ) , toluene / H O,
100 ° C, 24 h. (c) Dimethyl malonate, NaH dry THF, 0 ° C.
2.2. Enzyme activities and In vitro results
The inhibitory enzyme activities against hexokinase II (HK-II) expressed as IC50 values (Table 1).
Table 1. The result of enzyme activity
Compounds
IC50 (µM)
7.47
0.40
0.29
0.27
1.86
1.01
0.99
2.15
2.53
5.70
16.8
38.1
14.0
27.1
MJ
Compound 1
Compound 2
Compound 3
Compound 5
Compound 6
Compound 7
Compound 9
Compound 12
Compound 13
Compound 14
Compound 17
Compound 18
Compound 21
The results were expressed as the mean of duplicates ± SD.

Based on the IC50values of HK-II enzyme inhibition, compounds that demonstrates less than 1 μM IC50 activity are listed as
3
> 2 > 1 > 7 > 6. The order of inhibitory activity was established taking into account inorganic phosphate release (see Table 1).
Methyl jasmonate used as reference standard. Preliminary enzyme inhibition studies revealed that we have identified handful
of novel MJ analogues much more potent than MJ itself.
To evaluate the effecs of these novel analogues on the viability of A549 (lung cancer) cell line, cells were treated with different
novel compounds and among all tested, compound-3 derivatives were found extremely potent when compared to commercial
HK-II inhibitor Methyl Jasmonate (MJ) (Figure 2a; p<0.0001). Additionally, compound 3 showed cytotoxic effects on A549 cell
line with significant decrease in cell viability to 0.36% (IC50 values: 4.250mM) ( see Figure 2b).
a.
b.
Figure 2. Cytotoxic activity of novel MJ analogues on A549 lung cancer cell line.
The cells were treated with increased doses (1-10mM) of novel compounds and Methyl Jasmonate (MJ) as a reference
standard. Control wells were treated with equal concentrations of DMSO solvent. a) After 24h of treatment, cells were applied
to viability assay. According to measurements, cell viability was decreased to 0.36% in 5mM compound 3 treated cells. Plots
indicate relative IC50 values of compound 3. b) compound 3 treated A549 cell line. The results were expressed as the mean ±
SD from three independent experiments ( p*** <0.0001).
A549 cells were treated with all different novel HK-II inhibitors and their effects on cell viability expressed as IC50 values, are
summarized in Table 2.
Table 2. The IC50 values of novel molecules in A549 cell line
The Results of A549 cell line
Compounds
IC50 Values

MJ
6.383 mM
4.564 mM
4.25 mM
9.778 mM
7.115 mM
9.011 mM
10.88 mM
9.878 mM
ND
Compound 1
Compound 3
Compound 5
Compound 9
Compound 12
Compound 13
Compound 14
Compound 17
The results were expressed as the mean of duplicates ± SD.
Based on the IC50 values, compounds 1 and 3 demonstrated reasonably good activity when compared to MJ. The order of
cytotoxic activity was established considering the percentage of viable cells. Methyl jasmonate used as a reference standard.
According to cell viability assays, measurable cytotoxic activity of novel HK-II inhibitors on lung cancer cell line were recorded.
Then we further investigated the cytotoxicity of various HK-II inhibitors on SKOV-3 ovarian cancer cell line. Our findings showed
that among these novel inhibitors, compound 3 was the most potent HK-II inhibitor as compared to other compounds synthesized
in our laboratory. Highly significant cell death percentages were recorded in SKOV-3 as well as A549 cell lines upon treatment
with 5mM compound 3 derivatives for 24h (Figure 3a; p<0.0001). Additionaly, IC50 values of compound 3 treated SKOV-3 cell
line was calculated as 1.772 mM. This data indicates compound 3 is more potent than MJ and any other analogues on SKOV-
3
ovarian cancer cell line (Figure 3b).
a.
b.
Figure 3. Cytotoxic activity of novel HK-II inhibitors on SKOV-3 ovarian cancer cell line.

The cells were treated with increased doses (1-10mM) of novel HK-II inhibitors and Methyl Jasmonate (MJ) as a reference
standard. Control wells were treated with equal concentrations of DMSO solvent. a) After 24h of treatment, cells were applied
to viability assay. According to measurements, cell viability was decreased to 0.28% in 5mM compound 3 treated cells. Plots
indicate relative IC50 values of b) compound 3 treated with SKOV-3 cell line. The results were expressed as the mean ± SD from
triple replicates ( p*** <0.0001).
SKOV-3 cells treated with the different HK-II inhibitors and their cytotoxic effects expressed as IC50 values, were summarized
in Table 3.
Table 3. The IC50 values of novel molecules in SKOV-3 cell line
The Results of SKOV-3 cell line
Compounds
MJ
IC50 Values
4.17 mM
Compound 1
6.077 mM
Compound 2
ND
Compound 3
1.772 mM
Compound 5
ND
Compound 6
Compound 9
ND
5.758 mM
Compound 12
Compound13
5.82 mM
ND
Compound 14
Compound 17
7.68 mM
ND
The results were expressed as the mean of duplicates ± SD.
According to the IC50 values, compound 3 proved to more potent on SKOV-3 cell line after 24 h exposure. The order of
cytotoxic activity was established considering the percentage of viable cells. Methyl jasmonate used as a reference standard.
The cell viability results evidently show that there might be some cell specificity of these novel compounds againist different
cancer cell lines.
Furthermore, effects of HK-II inhibition on HK-II protein expression levels and biochemical verification of cell death were
studied by western blot analysis.
According to our results, HK-II expression was increased when treated with novel HK-II inhibitor compound 3 (Figure 4a).
This is most likely due to a cellular defense to compansate HK-II activity by increasing its protein expression levels. Since the
expression level is not linked to inhibitory effect of HK-II inhibitors, the cell death is inevitable once the cells recieve the inhibitory
signals.
Poly-ADP-ribose polymerase (PARP) cleavage is one of the best biochemical marker of cell death and PARP is cleaved at the
very downstream of apoptotic cascade, thus indicating the last stages of cell death at which DNA damage occurs [9], [10].
Therefore, we futher studied compound 3 triggered cell death at the protein level and showed the presence of cleaved PARP
as well as decresed levels of its precursor (PARP). Beside that, we did not detect cleaved PARP in control cells treated with
equal concentrations of DMSO as expected (Figure 4b).
a.

b.

Figure 4. HK-II protein expression and PARP cleavage levels of Compound 3 (C3) treated A549 lung cancer cell line. The cells
were treated with Compound 3 (IC50=4.25 mM) and control medium (DMSO solvent control) for 24h to detect expression of
HK-II and cell death proteins. Protein expression levels were determined by ImageJ and normalized to β-actin levels. a)
Treatment of A549 cell line with HK-II inhibitor compound 3 resulted in significantly increased HK-II expressions as compared
to control treatment, b) Compound 3 treated cells showed decreased PARP and increased c-PARP levels significantly as
#
compared to control. The results were expressed as the mean ± SD from three independent experiments. (p <0.01; p* <0.005;
and p** <0.0005).
Based on the literature, HK-II shows its activity in two ways: either catalyzing the reaction of 6-glucose phosphate formation
from glucose in glycolysis or binding VDAC on mitochondria [12,13,14,15]. Inhibition of both actions results in cell death. HK-II
inhibitors such as 2DG prevent the catalytic activity of the enyzme while MJ acts through the interruption of its mitochondrial
interaction [16]. Therefore, MJ analogues produced in our laboratory as HK-II inhibitors are predicted to demonstrate the same
mechanism of action with MJ. For this aim, we investigated the inhibitory effect of Compound 3 (C3) on HK-II and VDAC
interaction via immunoprecipitation of HK-II followed by western blotting of VDAC.
Figure 5. HK-II bound VDAC expression in mitochondrial protein extracts of C3 and DMSO (control) treated A549 lung cancer
cell line. The cells were treated with C3 (IC50=4.25 mM) and control medium (DMSO solvent control) for 24h and mitochondrial
proteins were extracted. To detect HK-II activity on VDAC binding, HK-II was immunoprecipitated (IP) and HK-II bound VDAC
expressions were detected by immunoblotting (IB). A549 cells treated with C3; MJ analogue showed decreased expression of
HK-II bound VDAC.
Based on our results, we identified more potent HK-II inhibitor (ex: C3) than MJ itself [11]. We also determined that the most
potent MJ analogue C3 acts by disposing VDAC and HK-II interaction on mitochondrial membrane; therefore induces cell death.
Even though MJ by itself is not reported for its enzymatic inhibitory role on HK-II, our enzyme inhibition assay suggests that the
novel MJ analogues can be potential candidates for HK-II inhibition. In order to fully undertsand the mechanism of action on
inhibiton of HK-II catalytic activity more investigations need to be conducted to clarify their behaviour. These studies suggest
that newly syntesized MJ analogues may have future potential as anti-cancer agents. Therefore, we will continue to pursue
additional in vitro experiments and in vivo efficacy studies of these novel analogues which will be reported on due course.
3. Experimental section
3.1. General
Anhydrous solvents and all reagents were purchased from Sigma-Aldrich, Merck and Alfa Aesar. Proton nuclear magnetic
1
resonance ( H NMR) spectra were recorded at Varian NMR (600, 500, 150 and 160 MHz, respectively). Chemical shifts were
1
reported in ppm downfield from tetramethylsilane (TMS) for proton and carbon. For H NMR spectra, chemical shifts are reported
in parts per million (ppm) and are reported relative to residual non-deuterated solvent signals. Coupling constants are reported

in hertz (Hz). The following abbreviations (or a combination, thereof) are used to describe splitting patterns: s, singlet; d, doublet;
t, triplet; q, quartet; quint, quintet; m, multiplet; comp, overlapping multiplets of non-magnetically equivalent protons; br, broad.
The mass spectra were obtained using a Shimadzu LC-MS 2040C equipped by Electrospray Source (ESI) operating in both
positive and negative ions. The solvents used in MS measures were methanol (Chromasolv grade), purchased from Sigma-
2
Aldrich and mQ water 18 MΩ, obtained from Millipore's Simplicity system. For the ionization, 5mM ammonium acetate in H O
was used in LC-MS. Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel F-254 plates.Coloumn
chromatography purifications were performed on Merck Silica gel 60 (0.063-0.200 mm ASTM) as the stationary phase. TLC
plates were visualized with UV light an also stained with ninhydrin, anisaldehyde or permanganat.
3.2. Chemical Synthesis
Synthesis of Jasmonic Acid; To methyl jasmonate (224.3 mg, 1 mmol) in 5 mL THF and 5 mL water was added LiOH.H
2
O (84
mg, 2 mmol). The reaction mixture was stirred at rt for 1 h and then diluted with 30 mL water. The aqueous layer was washed
by hexanes (30 mL), acidified by 1N HCl and extracted with EtOAc. Evaporation of the solvent after dried over MgSO
product as light yellow oil [8].
4
gave the
Compound 1: (3-((3-methyl-1,2,4-oxadiazol-5-yl)methyl)-2-(pent-2-en-1-yl)cyclopentanone; (a) 1 mL of a 0.5 M jasmonic acid
solution (0.5 mmol) in DMF was added to reaction flask, followed by addition of 0.55 mL of 1.0 M solution (0.55 mmol) of DCC
in DMF. After mixing for 30 min, 1.0 mL of a 0.55 M acetamide oxime solution (0.55 mmol) in DMF was added and the resulting
solution was mixed for 4 h at 25°C. (b) A further 0.55 mL of 1.0 M DCC (0.55 mmol) in DMF was added and the reaction mixtures
were heated to 115 °C for 6 h to effect cyclodehydration. After cooling to room temperature, 7 mL of DCM was added to each
vessel, followed by 3 mL of water. After 10 min of agitation, DCM layer washed with 1 x 3 mL H
sat. NaHCO , and 1 x 3 mL brine. The organic layers were dried with MgSO and filtrated. Purified by coloumn chromatography
) δ 5.49 – 5.42 (m, 1H), 5.28 – 5.21 (m, 1H), 3.22 (dd, J = 15.2,
.5 Hz, 1H), 2.87 (dd, J = 15.3, 9.1 Hz, 1H), 2.43 – 2.38 (m, 3H), 2.38 (s, 3H), 2.37 – 2.33 (m, 1H), 2.21 – 2.15 (m, 1H), 2.15 –
2
0, 1 x 3 mL 1 N HCI, 1 x 3 mL
3
4
1
(
3:2 EA:Hxn) [17]. Yellow oil (56%). H NMR (500 MHz, CDCl
3
4
2
13
.08 (m, 1H), 2.08 – 2.00 (m, 2H), 2.00 – 1.94 (m, 1H), 1.59 – 1.50 (m, 1H), 0.94 (t, J = 7.5 Hz, 3H). C NMR (125 MHz, CDCl
), 30.94 (CH
O2 requires 249.15.
3
)
2
),
δ 218.09 (C=O), 177.69 (CqAr), 167.17 (CqAr), 134.43 (CH), 124.62 (CH), 53.88 (CH), 39.30(CH), 37.61 (CH
6.94 (CH ), 25.52 (CH ), 20.60 (CH ), 14.09 (CH ), 11.54 (CH ). LCMS m/z :[M+H], found 248.32. C14
2
2
2
2
2
3
3
20 2
H N
Compound 2: 2-(pent-2-en-1-yl)-3-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)cyclopentanone; (a) To a mixture of the jasmonic acid
1 mmol) in acetone (4 mL) was added DCC (1.1 mmol). The reaction mixture was stirred at room temperature for 30 min, then
(
the benzamide oxime (1 mmol) was added. The resulting mixture was stirred at room temperature for 12 h. Acetone was
evaporated at reduced pressure and to the residue was added water (20 mL). The mixture was concentrated to give a residue.
(b) To a solution of O-acylbenzamidoxime 1 (1 mmol) in DMSO (2–3 mL) was added KOH (1 mmol). The reaction mixture was
stirred at room temperature for 10– 20 min (TLC or precipitation of the product). The reaction mixture was diluted with 30 ml of
cold water. The resulting was washed with water (30 mL). Purified by coloumn chromatography with 2:3 (EA:Hxn) solvent
1
mixture [18]. White oil (60%). H NMR (500 MHz, CDCl
3
) δ 8.02 – 7.99 (m, 2H), 7.45 – 7.39 (m, 3H), 5.45 – 5.38 (m, 1H), 5.25
–
2
0
1
5.18 (m, 1H), 3.26 (dd, J = 15.2, 4.7 Hz, 1H), 2.92 (dd, J = 15.2, 9.0 Hz, 1H), 2.47 – 2.39 (m, 1H), 2.38 – 2.34 (m, J = 3.4 Hz,
H), 2.34 – 2.30 (m, 1H), 2.22 – 2.15 (m, 1H), 2.07 (ddd, J = 18.9, 11.3, 8.9 Hz, 1H), 2.03 – 1.95 (m, 3H), 1.58 – 1.52 (m, 1H),
13
.89 (t, J = 7.5 Hz, 3H). C NMR (126 MHz, CDCl
28.88 (CHArX2), 127.43 (CHArX2), 126.74 (CqAr), 124.66 (CH), 53.94 (CH), 39.40 (CH), 37.65 (CH
), 25.62 (CH ), 20.65 (CH ), 14.11 (CH ). LCMS m/z : [M+H], found 310.39. C19 requires 311.05.
3
) δ 218.15 (C=O), 177.98 (CqAr), 168.40 (CqAr), 134.49 (CH), 131.24 (CHAr),
2
), 31.13 (CH ), 27.01
2
(CH
2
2
2
3
22 2 2
H N O
Compound 3: 3-((3-methyl-1,2,4-oxadiazol-5-yl)methyl)-2-(pent-2-en-1-yl)cyclopentanol; Added sodium borohydride (1.5 eq) to
a solution of 1 (1 eq) in methanol (10 mL) at 0 °C. The reaction mixture was stirred for 3 hours. Quenched the reaction mixture
with of 1M HCl. The mixture was diluted with ethyl acetate. Removed the organic layer, the aqueous layer. The organic layers
1
were dried with MgSO
4
and filtrated. Purified by coloumn chromatography. H NMR (500 MHz, CDCl
3
) δ 5.50 – 5.41 (m, 1H),
5
.41 – 5.32 (m, 1H), 3.96 – 3.89 (m, 1H), 3.05 (dd, J = 15.3, 5.3 Hz, 1H), 2.85 (dd, J = 15.3, 8.7 Hz, 1H), 2.36 (s, J = 8.4 Hz,
13
3
H), 2.15 – 2.09 (m, 2H), 2.08 – 1.99 (m, 3H), 1.89 – 1.78 (m, 2H), 1.67 – 1.52 (m, 3H), 0.95 (t, J = 7.5 Hz, 3H). C NMR (126
) δ 178.84 (Car), 166.87 (Car), 133.74 (CH), 126.26 (CH), 78.26 (CH), 53.51 (CH), 41.32(CH), 33.28 (CH ), 31.74
), 30.21 (CH ), 28.85 (CH ), 20.62 (CH ), 14.19 (CH ), 11.51 (CH ). LCMS m/z : [M+H], found 251. C14 requires
50.34.
MHz, CDCl
CH
3
2
(
2
2
2
2
3
3
22 2 2
H N O
2
Compound 5: 3-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)-2-(pent-2-en-1-yl)cyclopentanone;
2 mL of hydrazine
monohydrate was placed in a 2 necked flask. Upon addition of jasmonic acid (1 mmol) dropwise, the system was stirred at 80 °
C. After 3 hours the reaction was terminated by LC-MS control. To avoid excess hydrazine, toluene was added to evaporate the
solvent in the rotary evaporator. LCMS m/z : [M+H]: 225. 2-(3-oxo-2-(pent-2-en-1-yl)cyclopentyl)acetohydrazide (1 mmol) was
dissolved in 5 mL of water and cooled to 0 °C. Then 4-chlorobenzoyl chloride (3 mmol) was carefully added dropwise. Acetonitrile
was added to the reaction mixture, which was solid while standing, and allowed to stir for 2 hours. After 2 hours, the solvent was
evaporated on a rotary evaporator. A white solid was obtained. The reaction was terminated by LC-MS. LCMS m/z: [M+H]: 363.
4
was dissolved in toluene. An excess amount of phosphorus (V) oxychloride was added and the mixture was stirred at 110°C
under reflux for 16 hours. The reaction was terminated by LC-MS control. The reaction mixture was cooled and neutralized by
adding dropwise to the NaHCO solution. Extraction was carried out with ethyl acetate and the organic phase was dried over
MgSO and then the solvent was evaporated in a rotary evaporator. It was purified by column chromatography with a 10: 1.5
EA: Hxn solution mixture [19]. H NMR (500 MHz, CDCl
3
4
1
3
) δ 7.97 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 8.5 Hz, 2H), 5.52 – 5.42 (m,
H), 5.32 – 5.23 (m, 1H), 3.31 (dd, J = 15.4, 4.6 Hz, 1H), 2.96 (dd, J = 15.4, 9.0 Hz, 1H), 2.52 – 2.35 (m, 3H), 2.30 – 2.21 (m,
H), 2.15 (dd, J = 19.2, 10.6 Hz, 1H), 2.10 – 1.99 (m, 3H), 1.71 – 1.56 (m, 2H), 0.95 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz,
1
1

CDCl
Car), 53.97 (CH), 39.23 (CH), 37.61 (CH
found 345, C19 requires 344,84.
3
) δ 218.07 (C=O), 165.18 (Car), 164.14 (Car), 138.02(Car), 134.40 (CH), 129.47 (CH), 128.04 (CH), 124.68 (CH), 122.29
(
2
), 30.06 (CH ), 27.04 (CH ), 25.63 (CH ), 20.63 (CH ), 14.12 (CH). LCMS m/z : [M+H]
2
2
2
2
H
2 2
21ClN O
Compound 6: N-(2-morpholinoethyl)-2-(3-oxo-2-(pent-2-en-1-yl)cyclopentyl)acetamide; To a mixture of compound jasmonic
acid in EtOAc and triethylamine (TEA) (2 eq), was added N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate (HBTU) (1 eq). After stirring for 1 h at room temperature, 4-(2-aminoethyl)morpholine (2 eq) was added
and the reaction mixture was stirred for 12 h. The mixture was washed with water, dried, and concentrated to give a residue
1
which was purified by coloumn chromatography with 10:3 (EA:Hxn) solvent mixture [7]. Yellow oil (83%). H NMR (500 MHz,
CDCl
1
3
3
) δ 5.98 (b, 1H), 5.42 – 5.35 (m, 1H), 5.26 – 5.19 (m, 1H), 3.64 (t, 4H), 3.34 – 3.28 (m, 2H), 2.52 (dd, J = 14.2, 4.6 Hz,
H), 2.42 (t, J = 6.0 Hz, 2H), 2.41 – 2.38 (m, 4H), 2.32 – 2.25 (m, 4H), 2.20 – 2.13 (m, 1H), 2.11 – 2.05 (m, 1H), 2.04 – 1.96 (m,
H), 1.86 – 1.81 (m, 1H), 1.50 – 1.40 (m, 1H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl
) δ 171.14 (C=O), 133.92
x2), 57.11 (CH ), 54.14 (CH), 53.35 (CH x2), 41.25 (CH ), 38.64 (CH), 37.73 (CH ), 35.51 (CH ),
), 20.62 (CH ), 14.16 (CH ). LCMS m/z: [M+H], found 322.44. C18 requires 323.10.
3
(CH), 125.25 (CH), 66.85 (CH
2
2
2
2
2
2
27.19 (CH
2
), 25.64 (CH
2
2
3
30 2 3
H N O
Compound
7:
2-(3-oxo-2-(pent-2-en-1-yl)cyclopentyl)-N-(2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-
yl)acetamide; To a mixture of compound jasmonic acid in EtOAc and TEA (2 eq), was added HBTU (1 eq). After stirring for 1
h at room temperature, D-(+)-Glucosamine hydrochloride (2 eq) in 2 mL water was added and the reaction mixture was stirred
for 12 h. The mixture was washed with water, dried, and concentrated to give a residue. Purified by coloumn chromatography
1
with 10:2 (DCM:MeOH) solvent mixture [7]. Yellow oil (70%). H NMR (500 MHz, CD
3
OD) δ 5.47 – 5.40 (m, 1H), 5.36 – 5.27 (m,
1
H), 3.90 – 3.79 (m, 2H), 3.76 – 3.60 (m, 2H), 3.00 (s, 1H), 2.87 (s, 1H), 2.68 – 2.58 (m, 1H), 2.43 – 2.19 (m, 6H), 2.13 – 2.03
13
(
m, 3H), 2.02 – 1.95 (m, 1H), 1.65 – 1.52 (m, 1H), 1.36 – 1.28 (m, 1H), 0.97 (t, J = 7.5 Hz, 3H). C NMR (125 MHz, CD
20.76 (C=O), 173.33 (C=O), 133.25 (CH), 124.96 (CH), 95.80 (CH), 91.17 (CH), 76.61 (CH), 74.63 (CH), 71.65 (CH), 61.39
CH ), 54.04 (CH), 40.26 (CH ), 38.20 (CH), 37.23 (CH ), 26.38 (CH ), 24.81 (CH ), 20.13 (CH ), 13.12(CH ). LCMS m/z :
M+H], found 371.43. C18 requires 372.10.
3
OD) δ
2
(
[
2
2
2
2
2
2
3
H29NO
7
Compound 9: Methyl ((3-oxo-2-(pent-2-en-1-yl)cyclopentyl)methyl)carbamate; A dry flask containing jasmonic acid (1 mmol),
- (Dimethylamino) pyridine (1.1 mmol) and TEA (1.1 mmol) in toluene was stirred under nitrogen and heated to 110 ° C. The
4
diphenylphosphoryl azide (DPPA) (1.1 mmol) was then added dropwise at 110 ° C and left under reflux for 16 h. The reaction
was terminated by LC-MS control. After toluene was evaporated, it was extracted with ethyl acetate, washed with brine. The
organic phase was dried over Na
M+H]: 208. To a mechanically stirred suspension of NaOMe in MeOH at room temperature was added 8 (1 mmol) in MeOH.
After 6h later, the solvent was evaporated and acidified with concentrated HCl to a pH of 1. Then extracted with H O and ether.
The aqueous layer was separated and the combined organic layers were dried with MgSO , concentrated to yellow oil. Purified
2
SO4, then the solvent was evaporated. It was used without any purification. LCMS m/z :
[
2
4
1
by coloumn chromatography with 10:1.5 (EtOAc:HXN) solvent mixture. Yellow oil (45%). H NMR (500 MHz, CDCl
3
) δ 5.49 –
.42 (m, 1H), 5.27 – 5.18 (m, 1H), 4.86 (s, 1H), 3.66 (s, 3H), 3.46 – 3.35 (m, 1H), 3.29 – 3.16 (m, 1H), 2.46 – 2.28 (m, 3H), 2.14
2.01 (m, 5H), 1.92 – 1.83 (m, 1H), 1.57 – 1.45 (m, 1H), 0.95 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, CDCl
) δ 219.13 (C=O),
57.24 (C=O), 134.17 (CH), 125.07 (CH), 52.26 (CH ), 52.17 (CH), 44.25 (CH ), 41.82, 37.53 (CH ), 25.74 (CH ), 24.93 (CH ),
0.57 (CH ), 14.12 (CH ). LCMS m/z : [M+H] found 240.20, C13 requires 239.31.
5
–
1
2
3
3
2
2
2
2
2
3
H21NO
3
Compound 10: 3-(2-hydroxyethyl)-2-(pent-2-en-1-yl)cyclopentanone; Chlorotrimethylsilane (TMS) (1.25 eq) was added to a
solution of sodium iodide (1.25 eq) in dry acetonitrile (5 ml). A solution of the methyl jasmonate (1 mmol) and triethylamine (0.3
ml) was added, and the resulting mixture stirred at 40 °C for 1.5 h. The product was extracted with light petroleum, and
evaporated to give the silyl ether as an yellow oil. The silyl ether was dissolved in dry THF and added dropwise to a suspension
of lithium aluminium hydride (4 eq) in THF at 0 °C. The mixture was stirred at room temperature for 2 h. Work-up with water and
1
purified by coloumn chromatography. Light yellow oil (55%). H NMR (500 MHz, CDCl
3
) δ 5.46 – 5.39 (m, 1H), 5.29 – 5.22 (m,
1
–
1
H), 3.80 – 3.69 (m, 2H), 2.38 – 2.34 (m, 2H), 2.34 – 2.31 (m, 1H), 2.22 – 2.16 (m, 1H), 2.10 – 1.95 (m, J = 10.9 Hz, 5H), 1.88
1.80 (m, 1H), 1.56 – 1.51 (m, 1H), 1.46 – 1.41 (m, 1H), 0.95 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, CDCl
) δ 133.79 (CH),
25.33 (CH), 60.95 (CH ), 54.91 (CH), 38.09 (CH), 38.04 (CH ), 37.67 (CH ), 27.27 (CH ), 25.53 (CH ), 20.64 (CH ), 14.19
). LCMS m/z : [M+H], found 196.29. C12 requires 197.20.
3
2
2
2
2
2
2
(CH
3
20 2
H O
Compound 12: 2-(6-(pent-2-en-1-yl)-2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-5-yl)ethanol; To a mechanically stirred
suspension of NaOMe in toluene at room temperature was added 10 in toluene followed by ethyl formate added dropwise. The
mixture was stirred for 3-4 h and then acidified with concentrated HCl to a pH of 1. Then extracted with H
aqueous layer was separated and the combined organic layers were dried with MgSO , concentrated to yellow oil [20]. To a
stirred solution of 11 (1 eq) in MeOH (10 mL) was added phenyl hydrazine (1.1 eq), and the mixture was stirred for 1.5 h at
room temperature. Solvent was removed under reduced pressure, and the residue was dissolved in CH Cl (300 mL), washed
with H O, dried with Na , and concentrated to oil. Purified by coloumn chromatography with 1:1 (EA:Hxn) solvent mixture
21]. Silica Gel Chromatography (SGC) afforded the title compound described with NMR spectroscopy. H NMR (500 MHz,
2
O and ether. The
4
2
2
2
2 4
O
1
[
3
CDCl ) δ 7.57 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 7.8 Hz, 2H), 7.36 (s, J = 4.2 Hz, 1H), 7.26 (t, 1H), 5.39 – 5.31 (m, 1H), 5.19 – 5.12
(
2
1
m, 1H), 3.80 – 3.68 (m, 2H), 3.19 – 3.13 (m, 1H), 2.89 (dd, J = 14.5, 8.1 Hz, 1H), 2.80 (ddd, J = 14.5, 7.2, 4.3 Hz, 1H), 2.34 –
.27 (m, 2H), 2.21 – 2.12 (m, 1H), 1.95 – 1.76 (m, 4H), 0.85 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, CDCl
40.16 (Car), 135.03 (CH), 134.12 (CH), 129.18 (CH), 127.55 (Car), 126.20 (CH), 124.95 (CH), 120.55 (CH), 61.05 (CH
), 30.03 (CH ), 28.43 (CH ), 20.50 (CH ), 14.10 (CH ). LCMS m/z : [M+H], found 297.55,
) δ 149.35 (Car),
), 47.56
3
2
(CH), 45.08 (CH), 39.57 (CH
2
2
2
2
3
C
19 24 2
H N O requires 296,41.

Compound 13: 2-(3-oxo-2-(pent-2-en-1-yl)cyclopentyl)ethyl 3-methylisoxazole-5-carboxylate; 10 (1 mmol) was dissolved in
DMF and cooled to 0 °C. NaH( 1.25 mmol) was added under nitrogen to the mixture for alkoxide formation. Simultaneously, 3-
methylisoxazole-5-carboxylic acid (1 mmol) was dissolved in DMF and carbonyldiimidazole (1.2 mmol) was added and stirred
for activation of the acid for 30 min. After 30 min, CDI-activated 3-methylisoxazole-5-carboxylic acid was added dropwise with
stirring the alkoxide added under nitrogen. The reaction was allowed to warm to room temperature and stirred. The mixture was
stirred for 5 h and then acidified with concentrated HCl to a pH of 1. Then extracted ethyl acetate and H
2
O. The organic phase
was dried over MgSO and then the solvent was evaporated in a rotary evaporator. Purified by column chromatography with a
4
1
1
1
2
1
: 2 mixture of EA: Hxn solution. Light yellow oil (70%). H NMR (500 MHz, CDCl
3
) δ 6.75 (d, J = 2.9 Hz, 1H), 5.44 – 5.37 (m,
H), 5.24 – 5.17 (m, 1H), 4.42 (t, J = 6.7 Hz, 2H), 2.36 – 2.32 (m, 6H), 2.24 – 2.16 (m, 2H), 2.11 – 1.97 (m, 5H), 1.89 – 1.79 (m,
13
H), 0.93 – 0.89 (m, 3H). C NMR (126 MHz, CDCl
24.96 (CH), 110.10 (CH), 64.18 (CH ), 54.63 (CH), 38.30 (CH), 37.84 (CH
), 14.10 (CH ), 11.41 (CH ). LCMS m/z : [M+H] found 306.10, C17H23NO
4
3
) δ 219.18 (C=O), 160.40 (Car), 159.95 (C=O), 156.79 (Car), 133.91 (CH),
), 33.33 (CH ), 27.07 (CH ), 25.39 (CH ), 20.54
requires 305.37.
2
2
2
2
2
(CH
2
3
3
Compound 14: 2-(3-oxo-2-(pent-2-en-1-yl)cyclopentyl)ethyl thiazole-4-carboxylate; 10 (1 mmol) was dissolved in DMF and
cooled to 0 ° C. NaH ( 1.25 mmol) was added under nitrogen to the mixture for alkoxide formation. Simultaneously, 3-
methylisoxazole-5-carboxylic acid (1 mmol) was dissolved in DMF and carbonyldiimidazole (1.2 mmol) was added and stirred
for activation of the acid for 30 min. After 30 min, CDI-activated 3-methylisoxazole-5-carboxylic acid was added dropwise with
stirring the alkoxide added under nitrogen. The reaction was allowed to warm to room temperature and stirred. The mixture was
stirred for 5 h and then acidified with concentrated HCl to a pH of 1. Then extracted ethyl acetate and H
2
O. The organic phase
was dried over MgSO and then the solvent was evaporated in a rotary evaporator. Purified by column chromatography with a
4
1
2
1
1
3
: 1 mixture of EA: Hxn solution. Yellow oil (60%). H NMR (500 MHz, CDCl ) δ 8.86 (d, J = 2.1 Hz, 1H), 8.23 (d, J = 2.1 Hz,
H), 5.46 – 5.37 (m, 1H), 5.30 – 5.20 (m, 1H), 4.50 – 4.44 (m, 2H), 2.41 – 2.32 (m, 3H), 2.32 – 2.20 (m, 2H), 2.14 – 2.08 (m,
H), 2.08 – 1.99 (m, 3H), 1.92 – 1.84 (m, 1H), 1.80 – 1.71 (m, 1H), 1.56 – 1.45 (m, 1H), 0.92 (t, J = 7.5 Hz, 3H). 13C NMR (126
MHz, CDCl
CH ), 54.75 (CH), 38.39 (CH), 37.92 (CH
M+H] found 308.50. C16 S requires 307.41.
3
) δ 219.47 (C=O), 161.21 (C=O), 153.49 (CHar), 147.95 (Car), 133.89 (CHar), 127.31 (CH), 125.04 (CH), 63.66
(
[
2
2
), 33.61 (CH ), 27.17 (CH ), 25.42 (CH ), 20.57 (CH ), 14.14 (CH ). LCMS m/z :
2
2
2
2
3
H21NO
3
Compound 17: Methyl 2-(3-(1-benzyl-3-(2-methoxyphenyl)ureido)-2-(pent-2-en-1-yl)cyclopentyl)acetate; Methy jasmonate (1
eq) ve benzylamine (1 eq) 5-10 ml in acetonitrile was added to reaction flask. After mixing for 1-2 h, Na(OAc) BH (1.5 eq) was
3
added and the resulting solution was stirred over night. To compound of 7 in DCM was added 2-metoxyphenyl isocyanate (1
eq). The reaction mixture was controlled by LC-MS and TLC. Purified by coloumn chromatography with a 1:2 mixture of EA:
1
Hxn solution. Light yellow oil (70%). H NMR (500 MHz, CDCl3) δ 8.15 – 8.12 (m, 1H), 7.39 – 7.26 (m, 5H), 7.05 (s, 1H), 6.93
–
6.86 (m, 2H), 6.75 – 6.71 (m, 1H), 5.45 – 5.32 (m, 2H), 4.84 – 4.75 (m, 1H), 4.69 (d, J = 17.6 Hz, 1H), 4.50 (d, J = 17.3 Hz,
H), 3.65 (s, J = 4.5 Hz, 3H), 3.55 (s, 2H), 2.64 (dd, J = 15.1, 4.3 Hz, 1H), 2.27 – 2.20 (m, 1H), 2.20 – 1.93 (m, 8H), 1.86 – 1.72
1
(
m, 1H), 1.29 – 1.17 (m, 1H), 0.95 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 173.21 (C=O), 155.80 (C=O), 147.68 (Car),
1
1
2
38.08 (Car), 132.78 (CH), 129.13 (Car), 128.80 (CH), 127.39 (CH), 127.27 (CH), 126.36 (CH), 121.82 (CH), 121.07 (CH),
19.01 (CH), 109.82 (CH), 58.87, 55.49, 51.40, 49.36 (CH ), 47.04, 41.21, 40.45 (CH ), 30.18 (CH ), 29.14 (CH ), 28.07 (CH ),
0.76 (CH ), 14.19 (CH ). LCMS m/z : [M+H] found 465.15. C28 requires 464,60.
2
2
2
2
2
2
3
36 2 4
H N O
Compound 18:
yl)cyclopentane-3,1-diyl))diacetate; Methy jasmonate (2 eq) ve 4-(2-aminoethyl)morpholine (2 eq) 5-10 ml in acetonitrile was
added to reaction flask. After mixing for 1-2 h, Na(OAc) BH (3 eq) was added and the resulting solution was stirred over night.
To compound of 8 in DCM was added 1,4-diisocyanate (1 eq). The reaction mixture was controlled by LC-MS and TLC. Purified
Dimethyl 2,2'-((1,14-dimorpholino-4,11-dioxo-3,5,10,12-tetraazatetradecane-3,12-diyl)bis(2-(pent-2-en-1-
3
1
by coloumn chromatography with a 1: 2 mixture of EA: Hxn solution. Light yellow oil (65%). H NMR (500 MHz, CDCl
3
) δ 5.38
5.21 (m, 4H), 3.71 – 3.64 (m, 8H), 3.62 (d, J = 5.1 Hz, 6H), 3.44 – 3.05 (m, 9H), 2.61 – 2.39 (m, 14H), 2.19 – 1.81 (m, 18H),
.67 – 1.48 (m, 6H), 1.46 – 1.07 (m, 5H), 0.94 – 0.89 (m, 6H). 13C NMR (126 MHz, CDCl
) δ 173.38 (C=O), 173.21 (C=O),
59.86 (C=O), 159.78 (C=O), 132.96 (CH), 132.41 (CH), 127.61 (CH), 126.56 (CH), 66.90 (CH ), 60.65, 58.49 (CH), 54.14
), 46.92, 46.48, 41.29, 40.52, 40.47 (CH ), 39.55(CH ), 38.55, 30.14 (CH ), 29.35 (CH ), 28.86
), 28.43 (CH ), 28.41(CH ), 28.39 (CH ), 28.36 (CH ), 28.04 (CH ), 20.56 (CH ),
), 14.17(CH ). LCMS m/z [M+H] found 818.10. C44 requires 817.11.
–
1
1
3
2
(
(
1
CH), 54.08 (CH), 51.34 (CH
CH ), 28.54 (CH
4.20 (CH
2
2
2
2
2
2
2
2
2
2
2
2
), 27.14 (CH
2
), 20.67 (CH
2
2
3
3
76 6 8
H N O
®
Compound 21: Dimethyl 2-(3-oxo-2-phenylcyclopentyl)malonate; 2-Cyclopentenone (1 mmol) and OXONE (1.2 mmol) were
dissolved in dry DCM in a 2 necked flask. 2N HBr (2.2 mmol) was added and the solvent turned orange. TEA (2 mL) was added
carefully after stirring at room temperature for 4 hours. After stirring overnight at room temperature, the reaction mixture was
neutralized by pH control, extracted with ethyl acetate, and the organic phase was dried over MgSO
evaporated in a rotary evaporator [22]. Brown solid (30%).Phenyl boronic acid (1.5mmol), Na CO (3mmol) was added to a
stirred mixture under nitrogen by adding a 1/1 10mL toluene / water mixture. 19 was added via dissolution in toluene. Finally,
moles of 2% Pd (PPh were added and heated at 100 ° C under nitrogen. After 24 hours, it was cooled to room temperature.
Extraction was carried out with ethyl acetate and the organic phase was dried over MgSO and then the solvent was evaporated
in a rotary evaporator. Light yellow oil (30%). H NMR (500 MHz, DMSO) δ 8.10 (t, J = 2.8 Hz, 1H), 7.69 (d, J = 7.5 Hz, 2H),
4
and the solvent was
2
3
3 4
)
4
1
7
1
1
.36 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.3 Hz, 1H), 2.51 – 2.46 (m, 4H). 13C NMR (126 MHz, DMSO) δ 206.39 (C=O), 160.01 (CAr),
40.65 (Cq), 130.80 (Cq), 127.38 (CHArx2), 127.18 (CH), 125.80 (CHArx2), 34.57 (CH ), 25.00 (CH ). LC-MS m/z : [M+H] :
59. Dimethyl malonate (1.5 mmol) in a 2 necked flask was placed under nitrogen and dissolved in 10 mL dry THF and cooled
2
2
to 0 ° C. After 30 min NaH (3 mmol) was added rapidly under nitrogen and the reaction continued at 0 °C. After 1 hour 20 (1
mmol) was added to the reaction medium. The reaction was allowed to warm to room temperature and allowed to stir for 4
hours. The reaction was terminated by TLC and LC-MS control. Extraction was carried out with 1N HCl and DCM. The organic

phase was dried over MgSO
4
and then the solvent was evaporated in a rotary evaporator. The column was purified by column
1
chromatography with a 4:1 mixture of EA: Hxn solution. Colorless oil (70%). H NMR (500 MHz, CDCl
3
) δ 7.33 (t, J = 7.5 Hz,
H), 7.25 (t, J = 7.4 Hz, 1H), 7.09 (d, J = 7.2 Hz, 2H), 3.69 (s, J = 9.7 Hz, 3H), 3.49 (d, J = 7.0 Hz, 1H), 3.35 (s, 3H), 3.30 (d, J
12.2 Hz, 1H), 3.09 – 2.97 (m, 1H), 2.61 – 2.51 (m, 1H), 2.50 – 2.34 (m, 2H), 1.86 – 1.72 (m, 1H). C NMR (126 MHz, CDCl )
3
2
=
13
δ 215.74 (C=O), 168.16 (C=O), 168.07 (C=O), 136.77 (Car), 129.03(CHarx2), 128.65 (CHarx2), 127.38 (CHar), 59.60 (CH),
5
2
4.23 (CH), 52.47 (CH
90.31.
3 3 2 2 18 5
), 52.18 (CH ), 43.90 (CH), 37.88 (CH ), 25.06 (CH ). LCMS m/z : [M+H] found 291.20. C16H O requires
3
.2. Biological Activity Studies
.2.1. Hexokinase - II Enzyme Activity Assay
3
The Universal Enzyme Assay Kit (R & D, EA004) was used to determine the hexokinase II enzyme inhibition for novel molecules.
This kit quantitatively separates inorganic phosphate as it converts ADP to AMP via coupling phosphatase found in its structure.
The liberated inorganic phosphate complexes with the malachite green phosphate detection reagents. The resulting complex
ratio is proportional to the formation of AMP from ADP by phosphatase. For this reason, the rate of inorganic phosphate
production reflects the kinetics of the kinase reaction. Enzyme activity assay was applied according to manufacturer’s
instructions. Briefly, a substrate mixture was prepared by using 0.5 mM ATP and 25 mM glucose. The human hexokinase-2
enzyme (rhHK2) was prepared to be 7.5 ng / μL and the coupling phosphatase 4 enzyme to be 10 μg / mL. Respectively, 20 μL
of buffer, 20 μL of rhHK2, 2 μL of sample and 10 μL of cCoupling phosphatase 4 were added to the well and incubated for 10
min. And then, 30 μL of malachite green reagent A, 100 μL of ultrapure water, 30 μL of malachite green reagent B was added
and incubated for 20 minutes. After incubation, the absorbance was read at 620 nm and the results were calculated according
to the following equation.
Adjusted phosphate release *
Specific
(nmol) x (1000 pmol/nmol)
Activity(pmol/min/µg)=
Incubation time (min) x amount of
enzyme (µg) x coupling rate**
*
Derived from the linear curve of the phosphate standard and adjusted to the control.
* The coupling ratio in specified conditions is 0.475.
*
3.2.2. Cell Viability Assay
The human lung carcinoma cell line A549 (ATCC # CCL-185) and the human ovarian cancer cell line SKOV-3 (ATCC # HTB-
7
1
7) used in the experiments were obtained from ATCC (USA). A549 cells were cultured in RPMI (Gibco) medium containing
0% FBS (Gibco), 1% Penicillin / Streptomycin (Gibco) and 1% L-Glutamatine. The cells were incubated at 37 °C in a 5% CO2
incubator. While SKOV-3 cells were cultured in McCoy’s 5a Modified Medium (Gibco) at the same conditions. After achieving
4
confluence, 1.5 x10 cells/well seeded in 96-well black plates and treated with novel molecules for 24h at six different
concentrations (0.1-10 mM), and each concentration was prepared as triplicates. The cell viability assay Cell-Titer Glo
(Promega, Madison, WI, USA) was performed according to manufacturer’s instructions. MJ used as control and DMSO in all of
the concentrations was 2.5 %. Three independent experiments were performed. The luminescence signal was read on the
SpectraMax i3x Multi-Mode Detection Platform (Moleculer Devices, USA) to determine the percentage of viable cells. IC50 values
were determined by Graphpad Prism 7.
3.2.3. Mitochondrial Protein Extraction
5
A549 cancer cells were seeded in 6-well plates at a density of 2x10 cells/well and incubated in 5% CO
2
incubator. Next day,
the cells were treated with compound 3 with its IC50 dose for A549 cell line. After 24 hours, the medium was removed and
centrifuged. The cells were obtained from the wells in RIPA (Thermo Fischer) buffer with protease inhibitor (Roche, cOmplete
™
, Mini, EDTA-free Protease Inhibitor Cocktail). The cells were collected as lysate and incubated at +4°C on the shaker for 20
minutes. Next, the lysate was centrifuged and the supernatant was collected. The supernatant was then centrifuged at 5000
rpm for 10 minutes. After centrifuging, the supernatant was collected and centrifuged at 13,000 rpm for 30 minutes. The
supernatant was removed and the pellet was suspended in RIPA buffer. Protein amount was determined for each sample by
using nanodrop spectrophotometric protein concentration calculator (IMPLEN P330).
3.2.4. Immunoprecipitation
500 µg of protein samples of mitochondrial extraction were taken into separate tubes and 30 µL of beads (4% agarose, Protein
G Sepharose 4 Fast Flow, GE Healthcare) were added into the samples. The samples were incubated on the shaker at +4°C
for 1 hour. After that, the tubes were centrifuged at 300 rpm for 2 minutes and the supernatant was collected. 1 µg of Hexokinase-
2
+
antibody (Cell Signaling, Hexokinase II, C64G5) was added into the supernatant and the samples were kept on the shaker at
4°C for about 1 hour. Afterwards, 75 µL of beads were added to each sample and incubated overnight at +4°C on the shaker.

The next day the samples were centrifuged at 3000 rpm for 2 minutes and the supernatant was discarded. Laemmli buffer with
0% β-mercaptoethanol (Biorad) was added into the pellets and the samples were incubated for 5-10 minutes at 100°C. Then
western blot procedure was applied as explained below.
1
3.2.5. Western Blot
5
For total protein collection as whole cell lysate, 2x10 cells were seeded into each well of 6 well plates. Then the cells were
incubated at 37° C in 5% CO for 24 hours. The culture medium was discarded and cells were treated with proper amount of C3
2
according to IC50 values of each cell line. After 24 hours of treatment, protein lysates were collected using Ripa lysis buffer with
protease inhibitor and incubated at 4°C for 30 minutes on shaker. Then pelleted and protein samples in supernatant were
collected seperately. Total protein concentration measured by nanodrop spectrophotometric protein concentration calculator
(IMPLEN P330) and SDS treated samples were prepared by using Laemmli buffer (Biorad). Equal amounts of protein (25 μg)
were electrophoresed by loading into 4-12% polyacrylamide gel and the proteins were separated by their molecular weight. And
then proteins were transferred to polyvinylidene difluoride (PVDF) membranes by using the Trans-Blot TurboTransfer System
(BioRad). After that, blocking of membranes was performed by using 5% non-fat milk dissolved in TBST (137 mM NaCl, 20 mM
Tris, 0.1% Tween-20) for 1 hour at room temperature. Membranes were incubated overnight with primary monoclonal antibodies
at 4 °C at on a shaker. Antibodies used in the experiments were as follows: monoclonal rabbit Hexokinase-2 antibody (12885S,
cell signaling) (1:750); monoclonal mouse β-actin antibody (CST #3700s; Cell Signaling) (1:1000); and anti- poly-ADP-ribose
polymerase (PARP) (CST #9542) (1:2000). The following day, the membranes were washed 3 times for 5 minutes with TBST
and then incubated with HRP-conjugated secondary antibodies (anti-mouse CST#7076S; anti-rabbit CST#7074S) (anti-rabbit
1: 2500; anti-mouse 1: 2500) which dissolved in TBST containing 5% non-fat milk powder for 1 hour at room temperature.
Membranes were washed 3 times with TBST for 5 minutes. Membrane images were obtained using ECL HRP substrate (thermo
fisher, Pierce) by ChemiDoc MP System (Bio-Rad). Protein levels were analyzed using the ImageJ program and band intensities
were normalized according to β-actin blots.
3.2.6. Statistics
Data were analyzed by ANOVA Tukey test with SPSS 19.0 when comparing two groups. Data were expressed with the mean
#
of duplicates ± SD and the differences were determined as p <0.01; p* <0.005; p** <0.0005 and p*** <0.0001.
4. Conclusion
To our knowledge, the novel MJ analogues inhibit HK-II activity through VDAC detachment from mitochondria. Further studies
of these molecules will provide us their possible mechanism of action on the conversion of glucose to glucose 6-phosphate in
glycolysis pathway. Our next set of MJ analogues might shed light on developing new potent anti-cancer agents. This will guide
us in preparing more and multi potent, efficacious and soluble drug candidates. In conclusion, we have identified several
promising bioactive molecules more potent than MJ that irreversibly inhibit HK-II.
Acknowledgements: This project (215S890) is funded by TUBITAK. We kindly appreciate for their support. Authors OSI, and
BEY, received funding from TUBITAK as a stipend for this project (TUBİTAK project no 215S890). We thank Yasemin Yozgat
for her invaluable assistance in biological assays, Melike Aybala Guzel for proofreading our manuscript, and Ozan Topcu for
his technical support in IP experiments in this study.
1
Supplementary material and/or Additional information: Scanned H and APT NMR spectra of final compounds are provided
in the electronic supplementary information. Supplementary data to this article can be found online at https://doi.org/...
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Graphical Abstract
Synthesis of Novel Methyl Jasmonate Derivatives and Evaluation of Their
Biological Activity in Various Cancer Cell Lines
Bilgesu Onur Sucu, Ozgecan Savlug Ipek, Sukran Ozdatli Kurtulus, Busra Emine Yazici, Nihal Karakas
and Mustafa Guzel*
O
R2
O
O
O
R1
O
R3
O
O
O
O
MJ
O
R4
O
O
Graphical Abstract Figure. Structure of Novel Methyl Jasmonate Analogues
1
6


Corresponding author. Tel.: +90-507-735-9431; fax: +90-212-531-7555; e-mail: mguzel@medipol.edu.tr
Highlights:


Increased the biological activity by synthesizing novel MJ analogues
Identified promising bioactive molecules more potent than MJ that inhibit HK-II
New MJ analogues inhibit HK-II activity through VDAC detachment from mitochondria
Determined the most potent compound acts by disposing VDAC and HK-II interaction


1
7